Macrocyclic benzofuran and azabenzofuran compounds for the treatment of hepatitis C

ABSTRACT

Compounds of formula I, including their salts, as well as compositions and methods of using the compounds are set forth. The compounds have activity against hepatitis C virus (HCV) and may be useful in treating those infected with HCV.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority of U.S. Provisional ApplicationSer. No. 61/750,967 filed Jan. 10, 2013 which is herein incorporated byreference.

FIELD OF THE INVENTION

The invention relates to novel compounds, including their salts, whichhave activity against hepatitis C virus (HCV) and which are useful intreating those infected with HCV. The invention also relates tocompositions and methods of making and using these compounds.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is a major human pathogen, infecting anestimated 170 million persons worldwide—roughly five times the numberinfected by human immunodeficiency virus type 1. A substantial fractionof these HCV infected individuals develop serious progressive liverdisease, including cirrhosis and hepatocellular carcinoma (Lauer, G. M.;Walker, B. D. N. Engl. J. Med. 2001, 345, 41-52).

HCV is a positive-stranded RNA virus. Based on a comparison of thededuced amino acid sequence and the extensive similarity in the5′-untranslated region, HCV has been classified as a separate genus inthe Flaviviridae family. All members of the Flaviviridae family haveenveloped virions that contain a positive stranded RNA genome encodingall known virus-specific proteins via translation of a single,uninterrupted, open reading frame.

Considerable heterogeneity is found within the nucleotide and encodedamino acid sequence throughout the HCV genome. At least six majorgenotypes have been characterized, and more than 50 subtypes have beendescribed. The major genotypes of HCV differ in their distributionworldwide, and the clinical significance of the genetic heterogeneity ofHCV remains elusive despite numerous studies of the possible effect ofgenotypes on pathogenesis and therapy.

The single strand HCV RNA genome is approximately 9500 nucleotides inlength and has a single open reading frame (ORF) encoding a single largepolyprotein of about 3000 amino acids. In infected cells, thispolyprotein is cleaved at multiple sites by cellular and viral proteasesto produce the structural and non-structural (NS) proteins. In the caseof HCV, the generation of mature non-structural proteins (NS2, NS3,NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. Thefirst one is believed to be a metalloprotease and cleaves at the NS2-NS3junction; the second one is a serine protease contained within theN-terminal region of NS3 (also referred to as NS3 protease) and mediatesall the subsequent cleavages downstream of NS3, both in cis, at theNS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B,NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiplefunctions, acting as a cofactor for the NS3 protease and possiblyassisting in the membrane localization of NS3 and other viral replicasecomponents. The complex formation of the NS3 protein with NS4A seemsnecessary to the processing events, enhancing the proteolytic efficiencyat all of the sites. The NS3 protein also exhibits nucleosidetriphosphatase and RNA helicase activities. NS5B (also referred to asHCV polymerase) is a RNA-dependent RNA polymerase that is involved inthe replication of HCV. The HCV NS5B protein is described in “StructuralAnalysis of the Hepatitis C Virus RNA Polymerase in Complex withRibonucleotides (Bressanelli; S. et al., Journal of Virology 2002,3482-3492; and Defrancesco and Rice, Clinics in Liver Disease 2003, 7,211-242.

Currently, the most effective HCV therapy employs a combination ofalpha-interferon and ribavirin, leading to sustained efficacy in 40% ofpatients (Poynard, T. et al. Lancet 1998, 352, 1426-1432). Recentclinical results demonstrate that pegylated alpha-interferon is superiorto unmodified alpha-interferon as monotherapy (Zeuzem, S. et al. N.Engl. J. Med. 2000, 343, 1666-1672). However, even with experimentaltherapeutic regimens involving combinations of pegylatedalpha-interferon and ribavirin, a substantial fraction of patients donot have a sustained reduction in viral load. Thus, there is a clear andimportant need to develop effective therapeutics for treatment of HCVinfection.

HCV-796, an HCV NS5B inhibitor, has shown an ability to reduce HCV RNAlevels in patients. The viral RNA levels decreased transiently and thenrebounded during dosing when treatment was with the compound as a singleagent but levels dropped more robustly when combined with the standardof care which is a form of interferon and ribavirin. The development ofthis compound was suspended due to hepatic toxicity observed duringextended dosing of the combination regimens. U.S. Pat. No. 7,265,152 andthe corresponding PCT patent application WO2004/041201 describecompounds of the HCV-796 class. Other compounds have been disclosed; seefor example, WO2009/101022, as well as WO 2012/058125.

What is therefore needed in the art are additional compounds which arenovel and effective against hepatitis C. Additionally, these compoundsshould provide advantages for pharmaceutical uses, for example, withregard to one or more of their mechanism of action, binding, inhibitionefficacy, target selectivity, solubility, safety profiles, orbioavailability. Also needed are new formulations and methods oftreatment which utilize these compounds.

SUMMARY OF THE INVENTION

One aspect of the invention is a compound of formula I, includingpharmaceutically acceptable salts thereof:

whereinn, m are independently 0 or 1;R¹ is methyl;R² is phenyl that is independently substituted with 0-2 halo or methoxyor is para substituted with W—Ar¹;W is —O— or —NH—;Ar¹ is phenyl or para-halophenyl;R³ is hydrogen, halo, or alkyl;R⁴, R⁵, R⁶ are independently selected from hydrogen, halo, alkyl,haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, hydroxyalkyloxy,alkoxyalkyloxy, COOR¹¹ and CON(R¹²)(R¹³);R¹¹ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected fromhalo, hydroxyl, alkoxy, and haloalkoxy;R¹², R¹³ are each independently hydrogen, alkyl or cycloalkyl with 0-3substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; orR¹² and R¹³ can form a ring by joining two atoms, one from each of R¹²and R¹³;R¹⁴ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected fromhalo, hydroxyl, alkoxy, and haloalkoxy;R⁷ is hydrogen, alkyl, halo, N(R²¹)(R²²), or alkylsulfonyl;R²¹ and R²² are independently hydrogen, alkyl, hydroxyalkyl,alkoxyalkyl, alkylsulfonyl, or alkylsulfonylalkyl;or N(R²¹)(R²²) taken together is azetidinyl, pyrrolidinyl, piperidinyl,or piperazinyl, and is substituted with 0-2 substituents selected fromalkyl, hydroxyalkyl, and hydroxy;X, Y are independently selected from CR³¹R³², CO, O, NR³³, NCN, S, SO,and S(O)₂;R³¹, R³² are each independently alkyl or cycloalkyl, further substitutedwith 0-3 substituents; orR³¹ and R³² can form a ring by joining two atoms, one from each of R³¹and R³²;R³³ is hydrogen, alkyl, cycloalkyl, or S(O)₂R¹¹ with 0-3 substituentsselected from halo, haloalkoxy, OR¹¹, NR¹²R¹³, COOR¹¹, CONR¹²R¹³,S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³;Z is an alkylene or alkenylene chain containing 0-12 groups selectedfrom O, NR⁴¹, S, S(O), S(O)₂, C(O), C(O)O, C(O)NR⁴¹, C(S)NR⁴¹,OC(O)NR⁴¹, NR⁴¹C(O)NR⁴², and Ar², provided that any O or S atom does notdirectly bond to another O or S atom, such that ring A is 11-24membered; and further wherein the alkylene or alkenylene chain issubstituted with 0-6 substituents selected from the group of alkyl,cycloalkyl, halo, alkoxy, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³,COOR¹¹, coNR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, NR¹⁴CONR¹²R¹³ and Ar³;R⁴¹, R⁴² is hydrogen, alkyl or cycloalkyl with 0-3 substituents selectedfrom halo, haloalkoxy, OR¹¹, NR¹²R¹³, COOR¹¹, CONR¹²R¹³, S(O)₂R¹¹,S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³;Ar² is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and issubstituted with 0-3 substituents selected from cyano, halo, alkyl,cycloalkyl, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³, COOR¹¹,CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³;Ar³ is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and issubstituted with 0-3 substituents selected from cyano, halo, alkyl,cycloalkyl, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³,COOR¹¹CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³.

The invention also relates to pharmaceutical compositions comprising acompound of formula 1, including a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.

In addition, the invention provides one or more methods of treatinghepatitis C infection comprising administering a therapeuticallyeffective amount of a compound of formula I to a patient.

Also provided as part of the invention are one or more methods formaking the compounds of formula I.

The present invention is directed to these, as well as other importantends, hereinafter described.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Unless otherwise specifically set forth elsewhere in the application,the following terms may be used herein and shall have the followingmeanings: “Hydrogen” or “H” refers to hydrogen, including its isotopes,such as deuterium. “Halo” means fluoro, chloro, bromo, or iodo. “Alkyl”means a straight or branched alkyl group composed of 1 to 6 carbons.“Alkenyl” means a straight or branched alkyl group composed of 2 to 6carbons with at least one double bond. “Cycloalkyl” means a monocyclicring system composed of 3 to 7 carbons. “Hydroxyalkyl,” “alkoxy” andother terms with a substituted alkyl moiety include straight andbranched isomers composed of 1 to 6 carbon atoms for the alkyl moiety.“Halo” includes all halogenated isomers from monohalo substituted toperhalo substituted in substituents defined with halo, for example,“Haloalkyl” and “haloalkoxy”, “halophenyl”, “halophenoxy.” “Aryl” meansa monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12carbon atoms, or a bicyclic fused ring system wherein one or both of therings is a phenyl group. Bicyclic fused ring systems consist of a phenylgroup fused to a four- to six-membered aromatic or non-aromaticcarbocyclic ring. Representative examples of aryl groups include, butare not limited to, indanyl, indenyl, naphthyl, phenyl, andtetrahydronaphthyl. “Heteroaryl” means a 5 to 7 membered monocyclic or 8to 11 membered bicyclic aromatic ring system with 1-5 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Parentheticand multiparenthetic terms are intended to clarify bonding relationshipsto those skilled in the art. For example, a term such as ((R)alkyl)means an alkyl substituent further substituted with the substituent R.Substituents which are illustrated by chemical drawing to bond atvariable positions on a multiple ring system (for example a bicyclicring system) are intended to bond to the ring where they are drawn toappend.

The invention includes all pharmaceutically acceptable salt forms of thecompounds. Pharmaceutically acceptable salts are those in which thecounter ions do not contribute significantly to the physiologicalactivity or toxicity of the compounds and as such function aspharmacological equivalents. These salts can be made according to commonorganic techniques employing commercially available reagents. Someanionic salt forms include acetate, acistrate, besylate, bromide,camsylate, chloride, citrate, fumarate, glucouronate, hydrobromide,hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate,pamoate, phosphate, succinate, sulfate, tartrate, tosylate, andxinofoate. Some cationic salt forms include ammonium, aluminum,benzathine, bismuth, calcium, choline, diethylamine, diethanolamine,lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine,potassium, sodium, tromethamine, and zinc.

Some of the compounds of the invention possess asymmetric carbon atoms.The invention includes all stereoisomeric forms, including enantiomersand diastereomers as well as mixtures of stereoisomers such asracemates. Some stereoisomers can be made using methods known in theart. Stereoisomeric mixtures of the compounds and related intermediatescan be separated into individual isomers according to methods commonlyknown in the art. The use of wedges or hashes in the depictions ofmolecular structures in the following schemes and tables is intendedonly to indicate relative stereochemistry, and should not be interpretedas implying absolute stereochemical assignments.

The invention is intended to include all isotopes of atoms occurring inthe present compounds. Isotopes include those atoms having the sameatomic number but different mass numbers. By way of general example andwithout limitation, isotopes of hydrogen include deuterium and tritium.Isotopes of carbon include ¹³C and ¹⁴C. Isotopically-labeled compoundsof the invention can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed herein, using an appropriate isotopically-labeled reagent inplace of the non-labeled reagent otherwise employed. Such compounds mayhave a variety of potential uses, for example as standards and reagentsin determining biological activity. In the case of stable isotopes, suchcompounds may have the potential to favorably modify biological,pharmacological, or pharmacokinetic properties.

As set forth above, the invention is directed to one or more compoundsof formula I, including pharmaceutically acceptable salts thereof:

whereinn, m are independently 0 or 1;R¹ is methyl;R² is phenyl that is independently substituted with 0-2 halo or methoxyor is para substituted with W—Ar¹;W is —O— or —NH—;Ar¹ is phenyl or para-halophenyl;R³ is hydrogen, halo, or alkyl;R⁴, R⁵, R⁶ are independently selected from hydrogen, halo, alkyl,haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, hydroxyalkyloxy,alkoxyalkyloxy, COOR¹¹ and CON(R¹²)(R¹³);R¹¹ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected fromhalo, hydroxyl, alkoxy, and haloalkoxy;R¹², R¹³ are each independently hydrogen, alkyl or cycloalkyl with 0-3substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; orR¹² and R¹³ can form a ring by joining two atoms, one from each of R¹²and R¹³;R¹⁴ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected fromhalo, hydroxyl, alkoxy, and haloalkoxy;R⁷ is hydrogen, alkyl, halo, N(R²¹)(R²²), or alkylsulfonyl;R²¹ and R²² are independently hydrogen, alkyl, hydroxyalkyl,alkoxyalkyl, alkylsulfonyl, or alkylsulfonylalkyl;or N(R²¹)(R²²) taken together is azetidinyl, pyrrolidinyl, piperidinyl,or piperazinyl, and is substituted with 0-2 substituents selected fromalkyl, hydroxyalkyl, and hydroxy;X, Y are independently selected from CR³¹R³², CO, O, NR³³, NCN, S, SO,and S(O)₂;R³¹, R³² are each independently alkyl or cycloalkyl, further substitutedwith 0-3 substituents; orR³¹ and R³² can form a ring by joining two atoms, one from each of R³¹and R³²;R³³ is hydrogen, alkyl, cycloalkyl, or S(O)₂R¹¹ with 0-3 substituentsselected from halo, haloalkoxy, OR¹¹, NR¹²R¹³, COOR¹¹, CONR¹²R¹³,S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³;Z is an alkylene or alkenylene chain containing 0-12 groups selectedfrom the group consisting of O, NR⁴¹, S, S(O), S(O)₂, C(O), C(O)O,C(O)NR⁴¹, C(S)NR⁴¹, O—C(O)NR⁴¹, NR⁴¹C(O)NR⁴², and Ar², provided that anyO or S atom does not directly bond to another O or S atom, such thatring A is 11-24 membered; and further wherein the alkylene or alkenylenechain is substituted with 0-6 substituents selected from alkyl,cycloalkyl, halo, alkoxy, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³,COOR¹¹, CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, NR¹⁴CONR¹²R¹³, and Ar³;R⁴¹, R⁴² is hydrogen, alkyl or cycloalkyl with 0-3 substituents selectedfrom halo, haloalkoxy, OR¹¹, NR¹²R¹³, COOR¹¹, CONR¹²R¹³, S(O)₂R¹¹,S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³;Ar² is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and issubstituted with 0-3 substituents selected from cyano, halo, alkyl,cycloalkyl, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³, COOR¹¹,CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³;Ar³ is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and issubstituted with 0-3 substituents selected from cyano, halo, alkyl,cycloalkyl, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³, COOR¹¹,CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³.

In a preferred embodiment of the invention, n and m are each 1.

It is also preferred that R² is phenyl substituted with halo, and evenmore preferably, fluoro or F.

Additionally, it is preferred that R³ and R⁴ and R⁶ and R⁷ are eachhydrogen or H.

In certain embodiments it is also preferred that R⁵ is CONR¹²R¹³.

It is also preferred that X and Y are each NR³³.

Further preferred is the embodiment wherein R³³ is hydrogen or isS(O)₂CH₃.

It is also preferred that A be equal to or greater than 12 members. Inother embodiments, it is preferred that A be equal to or greater than 15members. In other embodiments, it is preferred that A be equal to orgreater than 17 members. In yet other embodiments, it is preferred thatA be equal to or greater than 19 members. By way of non-limitingexample, a compound of the invention wherein ring A has 15 members couldbe represented as follows (with ring numbers included for illustration):

Also preferred are compounds of formula I, including pharmaceuticallyacceptable salts thereof, which are selected from the group of:

Pharmaceutical Compositions and Methods of Treatment

The compounds according to the various embodiments herein set forthdemonstrate activity against HCV NS5B, and can be useful in treating HCVand HCV infection. Therefore, another aspect of the invention is acomposition comprising a compound of formula I, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.

Another aspect of the invention is a composition further comprising anadditional compound having anti-HCV activity.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is an interferon or a ribavirin. Another aspectof the invention is wherein the interferon is selected from interferonalpha 2B, pegylated interferon alpha, consensus interferon, interferonalpha 2A, interferon lambda, and lymphoblastoid interferon tau.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is a cyclosporin. Another aspect of theinvention is where the cyclosporin is cyclosporin A.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is selected from the group consisting ofinterleukin 2, interleukin 6, interleukin 12, a compound that enhancesthe development of a type 1 helper T cell response, interfering RNA,anti-sense RNA, Imiqimod, ribavirin, an inosine 5′-monophospatedehydrogenase inhibitor, amantadine, and rimantadine.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is effective to inhibit the function of atarget selected from HCV metalloprotease, HCV serine protease, HCVpolymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCVegress, HCV NS5A protein, IMPDH, and a nucleoside analog for thetreatment of an HCV infection.

Another aspect of the invention is a composition comprising a compoundof formula I, or a pharmaceutically acceptable salt thereof, apharmaceutically acceptable carrier, an interferon and ribavirin.

Another aspect of the invention is a method of inhibiting the functionof the HCV replicon comprising contacting the HCV replicon with acompound of formula I or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is a method of inhibiting the functionof the HCV NS5B protein comprising contacting the HCV NS5B protein witha compound of formula I or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is a method of treating an HCV infectionin a patient comprising administering to the patient a therapeuticallyeffective amount of a compound of formula I or a pharmaceuticallyacceptable salt thereof. In another embodiment the compound is effectiveto inhibit the function of the HCV replicon. In another embodiment thecompound is effective to inhibit the function of the HCV NS5B protein.

Another aspect of the invention is a method of treating an HCV infectionin a patient comprising administering to the patient a therapeuticallyeffective amount of a compound of formula I, or a pharmaceuticallyacceptable salt thereof, in conjunction with (prior to, after, orconcurrently) another compound having anti-HCV activity.

Another aspect of the invention is the method wherein the other compoundhaving anti-HCV activity is an interferon or a ribavirin.

Another aspect of the invention is the method where the interferon isselected from interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, interferon lambda, and lymphoblastoidinterferon tau.

Another aspect of the invention is the method where the other compoundhaving anti-HCV activity is a cyclosporin.

Another aspect of the invention is the method where the cyclosporin iscyclosporin A.

Another aspect of the invention is the method where the other compoundhaving anti-HCV activity is selected from interleukin 2, interleukin 6,interleukin 12, a compound that enhances the development of a type 1helper T cell response, interfering RNA, anti-sense RNA, Imiqimod,ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor,amantadine, and rimantadine.

Another aspect of the invention is the method wherein the other compoundhaving anti-HCV activity is effective to inhibit the function of atarget selected from the group consisting of HCV metalloprotease, HCVserine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCVentry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and anucleoside analog for the treatment of an HCV infection.

Another aspect of the invention is the method wherein the other compoundhaving anti-HCV activity is effective to inhibit the function of targetin the HCV life cycle other than the HCV NS5B protein.

“Therapeutically effective” means the amount of agent required toprovide a meaningful patient benefit as understood by practitioners inthe field of hepatitis and HCV infection.

“Patient” means a person infected with the HCV virus and suitable fortherapy as understood by practitioners in the field of hepatitis and HCVinfection.

“Treatment,” “therapy,” “regimen,” “HCV infection,” and related termsare used as understood by practitioners in the field of hepatitis andHCV infection.

The compounds of this invention are generally given as pharmaceuticalcompositions comprised of a therapeutically effective amount of acompound or its pharmaceutically acceptable salt and a pharmaceuticallyacceptable carrier and may contain conventional excipients.Pharmaceutically acceptable carriers are those conventionally knowncarriers having acceptable safety profiles. Compositions encompass allcommon solid and liquid forms including for example capsules, tablets,lozenges, and powders as well as liquid suspensions, syrups, elixers,and solutions. Compositions are made using common formulationtechniques, and conventional excipients (such as binding and wettingagents) and vehicles (such as water and alcohols) are generally used forcompositions. See, for example, Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa., 17th edition, 1985.

Solid compositions are normally formulated in dosage units andcompositions providing from about 1 to 1000 mg of the active ingredientper dose are preferred. Some examples of dosages are 1 mg, 10 mg, 100mg, 250 mg, 500 mg, and 1000 mg. Generally, other agents will be presentin a unit range similar to agents of that class used clinically.Typically, this is 0.25-1000 mg/unit.

Liquid compositions are usually in dosage unit ranges. Generally, theliquid composition will be in a unit dosage range of 1-100 mg/mL. Someexamples of dosages are 1 mg/mL, 10 mg/mL, 25 mg/mL, 50 mg/mL, and 100mg/mL. Generally, other agents will be present in a unit range similarto agents of that class used clinically. Typically, this is 1-100 mg/mL.

The invention encompasses all conventional modes of administration; oraland parenteral methods are preferred. Generally, the dosing regimen willbe similar to other agents used clinically. Typically, the daily dosewill be 1-100 mg/kg body weight daily. Generally, more compound isrequired orally and less parenterally. The specific dosing regimen,however, will be determined by a physician using sound medical judgment.

The invention also encompasses methods where the compound is given incombination therapy. That is, the compound can be used in conjunctionwith, but separately from, other agents useful in treating hepatitis andHCV infection. In these combination methods, the compound will generallybe given in a daily dose of 1-100 mg/kg body weight daily in conjunctionwith other agents. The other agents generally will be given in theamounts used therapeutically. The specific dosing regimen, however, willbe determined by a physician using sound medical judgment.

Some examples of compounds suitable for compositions and methods arelisted in Table 1.

TABLE 1 Type of Inhibitor or Brand Name Physiological Class TargetSource Company NIM811 Cyclophilin Inhibitor Novartis ZadaxinImmuno-modulator Sciclone Suvus Methylene blue Bioenvision Actilon TLR9agonist Coley (CPG10101) Batabulin (T67) Anticancer β-tubulin inhibitorTularik Inc., South San Francisco, CA ISIS 14803 Antiviral antisenseISIS Pharmaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc., NewYork, NY Summetrel Antiviral antiviral Endo Pharmaceuticals HoldingsInc., Chadds Ford, PA GS-9132 Antiviral HCV Inhibitor Achillion/Gilead(ACH-806) Pyrazolopyrimidine Antiviral HCV Inhibitors Arrow Therapeuticscompounds and Ltd. salts From WO- 2005047288 26 May 2005 LevovirinAntiviral IMPDH inhibitor Ribapharm Inc., Costa Mesa, CA MerimepodibAntiviral IMPDH inhibitor Vertex (VX-497) Pharmaceuticals Inc.,Cambridge, MA XTL-6865 Antiviral monoclonal antibody XTL (XTL-002)Biopharmaceuticals Ltd., Rehovot, Israel Telaprevir Antiviral NS3 serineprotease Vertex (VX-950, inhibitor Pharmaceuticals LY-570310) Inc.,Cambridge, MA/Eli Lilly and Co. Inc., Indianapolis, IN HCV-796 AntiviralNS5B Replicase Wyeth/Viropharma Inhibitor NM-283 Antiviral NS5BReplicase Idenix/Novartis Inhibitor GL-59728 Antiviral NS5B ReplicaseGene Labs/ Inhibitor Novartis GL-60667 Antiviral NS5B Replicase GeneLabs/ Inhibitor Novartis 2'C MeA Antiviral NS5B Replicase GileadInhibitor PSI 6130 Antiviral NS5B Replicase Roche Inhibitor R1626Antiviral NS5B Replicase Roche Inhibitor 2'C Methyl Antiviral NS5BReplicase Merck adenosine Inhibitor JTK-003 Antiviral RdRp inhibitorJapan Tobacco Inc., Tokyo, Japan Levovirin Antiviral ribavirin ICNPharmaceuticals, Costa Mesa, CA Ribavirin Antiviral ribavirinSchering-Plough Corporation, Kenilworth, NJ Viramidine AntiviralRibavirin Prodrug Ribapharm Inc., Costa Mesa, CA Heptazyme Antiviralribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO BILN-2061 Antiviralserine protease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim,Germany SCH 503034 Antiviral serine protease Schering Plough inhibitorZadazim Immune modulator Immune modulator SciClone Pharmaceuticals Inc.,San Mateo, CA Ceplene Immunomodulator immune modulator MaximPharmaceuticals Inc., San Diego, CA CellCept Immunosuppressant HCV IgGimmuno- F. Hoffmann-La suppressant Roche LTD, Basel, Switzerland CivacirImmunosuppressant HCV IgG immuno- Nabi suppressant BiopharmaceuticalsInc., Boca Raton, FL Albuferon-α Interferon albumin IFN-α2b Human GenomeSciences Inc., Rockville, MD Infergen A Interferon IFN InterMunealfacon-1 Pharmaceuticals Inc., Brisbane, CA Omega IFN Interferon IFN-ωIntarcia Therapeutics IFN-β and EMZ70 1 Interferon IFN-β and EMZ701Transition Therapeutics Inc., Ontario, Canada Rebif Interferon IFN-β1aSerono, Geneva, Switzerland Roferon A Interferon IFN-α2a F. Hoffmann-LaRoche LTD, Basel, Switzerland Intron A Interferon IFN-α2bSchering-Plough Corporation, Kenilworth, NJ Intron A and InterferonIFN-α2b/α1-thymosin RegeneRx Zadaxin Biopharma. Inc., Bethesda, MD/SciClone Pharmaceuticals Inc, San Mateo, CA Rebetron InterferonIFN-α2b/ribavirin Schering-Plough Corporation, Kenilworth, NJ ActimmuneInterferon INF-γ InterMune Inc., Brisbane, CA Interferon-β InterferonInterferon-β-1a Serono Multiferon Interferon Long lasting IFN Viragen/Valentis Wellferon Interferon Lympho-blastoid IFN- GlaxoSmithKline αn1plc, Uxbridge, UK Omniferon Interferon natural IFN-α Viragen Inc.,Plantation, FL Pegasys Interferon PEGylated IFN-α2a F. Hoffmann-La RocheLTD, Basel, Switzerland Pegasys and Interferon PEGylated IFN-α2a/ MaximCeplene immune modulator Pharmaceuticals Inc., San Diego, CA Pegasys andInterferon PEGylated IFN- F. Hoffmann-La Ribavirin α2a/ribavirin RocheLTD, Basel, Switzerland PEG-Intron Interferon PEGylated IFN-α2bSchering-Plough Corporation, Kenilworth, NJ PEG-Intron/ InterferonPEGylated IFN- Scheringng-Plough Ribavirin α2b/ribavirin Corporation,Kenilworth, NJ IP-501 Liver protection antifibrotic IndevusPharmaceuticals Inc., Lexington, MA IDN-6556 Liver protection caspaseinhibitor Idun Pharmaceuticals Inc., San Diego, CA ITMN-191 (R-7227)Antiviral serine protease InterMune inhibitor Pharmaceuticals Inc.,Brisbane, CA GL-59728 Antiviral NS5B Replicase Genelabs InhibitorANA-971 Antiviral TLR-7 agonist Anadys Boceprevir Antiviral serineprotease Schering Plough inhibitor TMS-435 Antiviral serine proteaseTibotec BVBA, inhibitor Mechelen, Belgium BI-201335 Antiviral serineprotease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim, GermanyMK-7009 Antiviral serine protease Merck inhibitor PF-00868554 Antiviralreplicase inhibitor Pfizer ANA598 Antiviral Non-Nucleoside Anadys NS5BPolymerase Pharmaceuticals, Inhibitor Inc., San Diego, CA, USA IDX375Antiviral Non-Nucleoside Idenix Replicase Inhibitor Pharmaceuticals,Cambridge, MA, USA BILB 1941 Antiviral NS5B Polymerase BoehringerInhibitor Ingelheim Canada Ltd R&D, Laval, QC, Canada PSI-7851 AntiviralNucleoside Pharmasset, Polymerase Inhibitor Princeton, NJ, USA PSI-7977Antiviral Nucleotide NS5B Pharmasset, Polymerase Inhibitor Princeton,NJ, USA VCH-759 Antiviral NS5B Polymerase ViroChem Pharma InhibitorVCH-916 Antiviral NS5B Polymerase ViroChem Pharma Inhibitor GS-9190Antiviral NS5B Polymerase Gilead Inhibitor Peg-interferon AntiviralInterferon ZymoGenetics/ lambda Bristol-Myers Squibb

Synthesis Methods

The compounds may be made by methods known in the art, including thosedescribed below. Some reagents and intermediates are known in the art.Other reagents and intermediates can be made by methods known in the artusing commercially available materials. The variables (e.g. numbered “R”substituents) used to describe the synthesis of the compounds areintended only to illustrate how to make and are not to be confused withvariables used in the claims or in other sections of the specification.Abbreviations used within the schemes generally follow conventions usedin the art.

Abbreviations used in the schemes generally follow conventions used inthe art. Chemical abbreviations used in the specification and examplesare defined as follows: “NaHMDS” for sodium bis(trimethylsilyl)amide;“DMF” for N,N-dimethylformamide; “MeOH” for methanol; “NBS” forN-bromosuccinimide; “Ar” for aryl; “TFA” for trifluoroacetic acid; “LAH”for lithium aluminum hydride; “DMSO” for dimethylsulfoxide; “h” forhours; “rt” for room temperature or retention time (context willdictate); “min” for minutes; “EtOAc” for ethyl acetate; “THF” fortetrahydrofuran; “EDTA” for ethylenediaminetetraacetic acid; “Et₂O” fordiethyl ether; “DMAP” for 4-dimethylaminopyridine; “DCE” for1,2-dichloroethane; “ACN” for acetonitrile; “DME” for1,2-dimethoxyethane; “HOBt” for 1-hydroxybenzotriazole hydrate; “DIEA”for diisopropylethylamine.

For the section of compounds in the 0000 series all LiquidChromatography (LC) data were recorded on a Shimadzu LC-10AS or LC-20ASliquid chromotograph using a SPD-10AV or SPD-20A UV-Vis detector andMass Spectrometry (MS) data were determined with a Micromass Platformfor LC in electrospray mode.

HPLC Method (i.e., Compound Isolation).

Compounds purified by preparative HPLC were diluted in methanol (1.2 mL)and purified using a Shimadzu LC-8A or LC-10A or Dionex APS-3000 orWaters Acquity™ automated preparative HPLC system.

EXAMPLES Preparation of Intermediates, Compound 3 and Compound 4

Step 1: To a mixture of2-(4-fluorophenyl)-3-(methylcarbamoyl)-6-nitrobenzofuran-5-yltrifluoromethanesulfonate (400 mg),(3-(tert-butylcarbamoyl)-5-nitrophenyl)boronic acid (345 mg) and Cs₂CO₃(846 mg) in dioxane (8 mL) and water (1.5 mL) was addedtetrakis(triphenylphosphine)palladium(0) (150 mg). The mixture wasflushed with nitrogen and then heated at 99° C. for 16 hours. Themixture was diluted with water and extracted with EtOAc (2×100 mL). Theorganic layers were combined, washed with brine, dried over MgSO₄ andconcentrated. The residue was purified by silica gel column(hexanes/EtOAc=3:1) to give5-(3-(tert-butylcarbamoyl)-5-nitrophenyl)-2-(4-fluorophenyl)-N-methyl-6-nitrobenzofuran-3-carboxamide2 (350 mg).

Compound 2 MS (2M + H)⁺ Calcd. 1069.3 MS (2M + H)⁺ Observ. 1069.2Retention Time 1.88 minutes LC Condition Solvent A 5% ACN:95% Water:10mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium ActetateSolvent % B   0 Final % B  100 Gradient Time 2 min Flow Rate 1 mL/minWavelength  220 Solvent Pair Acn:Water:Ammonium Actetate ColumnPhenomenex LUNA C18, 30 × 2, 3 uStep 2: To a solution of5-(3-(tert-butylcarbamoyl)-5-nitrophenyl)-2-(4-fluorophenyl)-N-methyl-6-nitrobenzofuran-3-carboxamide(350 mg) in MeOH (50 mL) was added palladium on carbon (69.7 mg). Themixture was stirred under hydrogen with a balloon for 16 hours. Pd/C wasremoved by filtration and the filtrate was concentrated to give6-amino-5-(3-amino-5-(tert-butylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide3 (200 mg).

Compound 3 MS (M + H)⁺ Calcd. 475.2 MS (M + H)⁺ Observ. 475.2 RetentionTime 0.80 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFASolvent B 10% Water-90% Methanol-0.1% TFA Start % B  50 Final % B 100Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Solvent PairWater-Methanol-TFA Column PHENOMENEX-LUNA 2.0 × 30 mm 3 umStep 3: To a solution of6-amino-5-(3-amino-5-(tert-butylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide(100 mg) and pyridine (0.170 mL) in CH₂Cl₂ (10 mL) was addedmethanesulfonyl chloride (72.4 mg) slowly at 0° C. The mixture wasstirred at room temperature for 16 hours. The reaction was quenched withaqueous NaHCO₃ and extracted with CH₂Cl₂. The organic layer wasconcentrated and the residue was purified by preparative HPLC to give5-(3-(tert-butylcarbamoyl)-5-(methylsulfonamido)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(methylsulfonamido)benzofuran-3-carboxamide4 (92 mg).

Compound 4 MS (M + H)⁺ Calcd. 631.2 MS (M + H)⁺ Observ. 631.3 RetentionTime 1.17 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFASolvent B 10% Water-90% Methanol-0.1% TFA Start % B  50 Final % B 100Gradient Time 2 min Flow Rate 1 mL/min Wavelength 220 Solvent PairWater-Methanol-TFA Column PHENOMENEX-LUNA 2.0 × 30 mm 3 um

General Procedure for the Preparation of Compounds 1001-1006

A mixture of5-(3-(tert-butylcarbamoyl)-5-(methylsulfonamido)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(methylsulfonamido)benzofuran-3-carboxamide4 (10 mg, 1 eq.), 1,4-dibromide (1 eq.) and K₂CO₃ (8.77 mg, 4 eq.) inacetonitrile (5 mL) and DMF (2 mL) was heated at 80° C. for 16 hours.All the solvents were removed under vacuum. The residue purified bypreparative HPLC.

LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10%Water-90% Methanol-0.1% TFA Start % B  30 Final % B 100 Gradient Time 2min Flow Rate 1 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFAColumn PHENOMENEX-LUNA 2.0 × 30 mm 3 um

MS MS Retention (M + H)⁺ (M + H)⁺ Time Cmpd# Structure Calcd. Observ.(min) 1001

685.2 685.4 1.60 1002

699.2 699.4 1.69 1003

713.2 713.4 1.83 1004

727.3 727.5 1.99 1005

741.3 741.5 2.05 1006

761.2 761.5 1.89

General Procedure for the Preparation of Compounds 1051-1058

To a solution of6-amino-5-(3-amino-5-(tert-butylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide3 (10 mg, 1 eq.) in CH₂Cl₂ (10 mL) was added diacylchloride ordicarbonochloridate or diisothiocyanate (0.8 eq.) and DIPEA (0.022 mL, 6eq.). The mixture was stirred at room temperature for 16 hours. All thesolvents were removed under vacuum. The residue was dissolved in 1.5 mLof DMF and purified by preparative HPLC.

LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA Solvent B 10%Water-90% Methanol-0.1% TFA Start % B  30 Final % B 100 Gradient Time 2min Flow Rate 1 mL/min Wavelength 220 Solvent Pair Water-Methanol-TFAColumn PHENOMENEX-LUNA 2.0 × 30 mm 3 um

MS MS Retention (M + H)⁺ (M + H)⁺ Time Cmpd# Structure Calcd. Observ.(min) 1051

585.2 585.4 1.64 1052

641.3 641.5 2.22 1053

669.3 669.5 2.29 1054

589.2 589.3 1.65 1055

633.2 633.4 1.81 1056

677.3 677.4 1.82 1057

647.2 647.4 1.90 1058

703.3 703.5 2.26

Preparation of Compound 2001

Step 1: Cs₂CO₃ (705 mg) and tetrakis(triphenylphosphine)palladium(0) (62mg) were added into a solution of2-(4-fluorophenyl)-3-(methylcarbamoyl)-6-nitrobenzofuran-5-yltrifluoromethanesulfonate(500 mg) and 3-(6-methyl-4,8-dioxo-1,3,6,2-dioxazaborocan-2-yl)benzoicacid (360 mg) in dioxane (9 mL) and water (3 mL). The reaction washeated at 90° C. for 4 hours. After being cooled to room temperature,the mixture was poured into ice water, which was adjusted to pH 3 by 2Naqueous HCl solution. The compound 101 formed as a brown solid and wasisolated via filtration.

Compound 101 MS (M + H)⁺ Calcd. 435.1 MS (M + H)⁺ Observ. 435.4Retention Time 1.37 minutes LC Condition Solvent A 5% ACN:95% Water:10mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium ActetateStart % B  0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/minWavelength 220 Solvent Pair ACN:Water:Ammonium Actetate ColumnPhenomenex LUNA C18, 30 × 2, 3 uStep 2: iPr₂NEt (89 mg) and was added into a solution of3-(2-(4-fluorophenyl)-3-(methylcarbamoyl)-6-nitrobenzofuran-5-yl)benzoicacid (150 mg), 2-amino-2-methylpropan-1-ol (37 mg) andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (171 mg) in DMF (2 mL). The reaction was stirred atroom temperature for 16 hours, before 20 mL of EtOAc was added. Theorganic layer was washed with water (2×10 mL), brine (5 mL) and driedover MgSO₄. After filtration, removal of solvents under vacuum affordeda residue which was used as was.

Compound 102 MS (M + H)⁺ Calcd. 506.2 MS (M + H)⁺ Observ. 506.6Retention Time 1.64 minutes LC Condition Solvent A 5% ACN:95% Water:10mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium ActetateStart % B  0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/minWavelength 220 Solvent Pair ACN:Water:Ammonium Actetate ColumnPhenomenex LUNA C18, 30 × 2, 3 uStep 3: To a solution of2-(4-fluorophenyl)-5-(3-((1-hydroxy-2-methylpropan-2-yl)carbamoyl)phenyl)-N-methyl-6-nitrobenzofuran-3-carboxamide(108 mg) in ethyl ether (4 mL), was added palladium on carbon (1.137mg). The mixture was stirred under hydrogen with a balloon for 16 hours.Pd/C was removed by filtration and the filtrate was concentrated to give6-amino-2-(4-fluorophenyl)-5-(3-((1-hydroxy-2-methylpropan-2-yl)carbamoyl)phenyl)-N-methylbenzofuran-3-carboxamide103 (95 mg).

Compound 103 MS (M + H)⁺ Calcd. 476.2 MS (M + H)⁺ Observ. 476.5Retention Time 1.52 minutes LC Condition Solvent A 5% ACN:95% Water:10mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium ActetateStart % B  0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/minWavelength 220 Solvent Pair ACN:Water:Ammonium Actetate ColumnPhenomenex LUNA C18, 30 × 2, 3 uStep 4: NaH (3.71 mg) was added into the solution of6-amino-2-(4-fluorophenyl)-5-(3-((1-hydroxy-2-methylpropan-2-yl)carbamoyl)phenyl)-N-methylbenzofuran-3-carboxamide(14.7 mg) and 3-chloro-2-(chloromethyl)prop-1-ene (3.86 mg) in DMF. Thereaction was stirred at room temperature for 24 hours, before beingquenched by MeOH. The compound 2001 was isolated by preparative HPLC.

Compound 2001 MS (M + H)⁺ Calcd. 528.2 MS (M + H)⁺ Observ. 528.6Retention Time 1.77 minutes LC Condition Solvent A 5% ACN:95% Water:10mM Ammonium Actetate Solvent B 95% ACN:5% Water:10 mM Ammonium ActetateStart % B  0 Final % B 100 Gradient Time 2 min Flow Rate 1 mL/minWavelength 220 Solvent Pair ACN:Water:Ammonium Actetate ColumnPhenomenex LUNA C18, 30 × 2, 3 u

Biological Methods

The compound demonstrated activity against HCV NS5B as determined in thefollowing HCV RdRp assays.

HCV NS5B RdRp Cloning, Expression, and Purification.

The cDNA encoding the NS5B protein of HCV, genotype 1b, was cloned intothe pET21a expression vector. The protein was expressed with an 18 aminoacid C-terminal truncation to enhance the solubility. The E. colicompetent cell line BL21(DE3) was used for expression of the protein.Cultures were grown at 37° C. for ˜4 hours until the cultures reached anoptical density of 2.0 at 600 nm. The cultures were cooled to 20° C. andinduced with 1 mM IPTG. Fresh ampicillin was added to a finalconcentration of 50 μg/mL and the cells were grown overnight at 20° C.

Cell pellets (3 L) were lysed for purification to yield 15-24 mgs ofpurified NS5B. The lysis buffer consisted of 20 mM Tris-HCl, pH 7.4, 500mM NaCl, 0.5% triton X-100, 1 mM DTT, 1 mM EDTA, 20% glycerol, 0.5 mg/mllysozyme, 10 mM MgCl₂, 15 μg/ml deoxyribonuclease I, and Complete™protease inhibitor tablets (Roche). After addition of the lysis buffer,frozen cell pellets were resuspended using a tissue homogenizer. Toreduce the viscosity of the sample, aliquots of the lysate weresonicated on ice using a microtip attached to a Branson sonicator. Thesonicated lysate was centrifuged at 100,000×g for 30 minutes at 4° C.and filtered through a 0.2 μm filter unit (Corning).

The protein was purified using two sequential chromatography steps:Heparin sepharose CL-6B and polyU sepharose 4B. The chromatographybuffers were identical to the lysis buffer but contained no lysozyme,deoxyribonuclease I, MgCl₂ or protease inhibitor and the NaClconcentration of the buffer was adjusted according to the requirementsfor charging the protein onto the column. Each column was eluted with aNaCl gradient which varied in length from 5-50 column volumes dependingon the column type. After the final chromatography step, the resultingpurity of the enzyme is >90% based on SDS-PAGE analysis. The enzyme wasaliquoted and stored at −80° C.

Standard HCV NS5B RdRp Enzyme Assay.

HCV RdRp genotype 1b assays were run in a final volume of 60 μl in 96well plates (Costar 3912). The assay buffer is composed of 20 mM Hepes,pH 7.5, 2.5 mM KCl, 2.5 mM MgCl₂, 1 mM DTT, 1.6 U RNAse inhibitor(Promega N2515), 0.1 mg/ml BSA (Promega R3961), and 2% glycerol. Allcompounds were serially diluted (3-fold) in DMSO and diluted further inwater such that the final concentration of DMSO in the assay was 2%. HCVRdRp genotype 1b enzyme was used at a final concentration of 28 nM. ApolyA template was used at 6 nM, and a biotinylated oligo-dT12 primerwas used at 180 nM final concentration. Template was obtainedcommercially (Amersham 27-4110). Biotinylated primer was prepared bySigma Genosys. ³H-UTP was used at 0.6 μCi (0.29 μM total UTP). Reactionswere initiated by the addition of enzyme, incubated at 30° C. for 60min, and stopped by adding 25 μL of 50 mM EDTA containing SPA beads (4μg/μL, Amersham RPNQ 0007). Plates were read on a Packard Top Count NXTafter >1 hr incubation at room temperature.

Modified HCV NS5B RdRp Enzyme Assay.

An on-bead solid phase homogeneous assay was also used to assess NS5Binhibitors (WangY-K, Rigat K, Roberts S, and Gao M (2006) Anal Biochem,359: 106-111). The assay is a modification of the standard assaydescribed above and was used in a 96-well or a 384-well format. Thebiotinylated oligo dT12 primer was captured on streptavidin-coupledbeads (SPA beads (GE, RPNQ0007) or imaging beads (GE, RPNQ0261) bymixing primer and beads in buffer and incubating at room temperature forthree hours. Unbound primer was removed after centrifugation. Theprimer-bound beads were resuspended in 3× reaction buffer (40 mM Hepesbuffer, pH 7.5, 7.5 mM MgCl₂, 7.5 mM KCl, dT primer coupled beads, polyA template, ³H-UTP, and RNAse inhibitor (Promega N2515). Compounds wereserially diluted 1:3 in DMSO and aliquoted into assay plates. Equalvolumes (20 μL for 96-well assay and 10 μL for 384-well assay) of water,3× reaction mix, and enzyme in 20 mM Hepes buffer, pH 7.5, 0.1 mg/ml BSAwere added to the diluted compound on the assay plate. Finalconcentration of components in 96-well assay: 0.36 nM template, 15 nMprimer, 0.43 μM (1 μCi)³H-UTP, 0.08 U/μL RNAse inhibitor, 7 nM NS5Benzyme, 0.033 mg mL BSA, and 2 μg/μL beads, 20 mM Hepes buffer, pH 7.5,2.5 mM MgCl₂, 2.5 mM KCl, 2% DMSO. Final concentration of components in384-well assay: 0.2 nM template, 15 nM primer, 0.29 μM ³H-UTP (0.3 μCi),0.08 U/μL RNAse inhibitor, 7 nM NS5B enzyme, 0.033 mg/mL BSA, and 0.33μg/μL beads, 20 mM Hepes buffer, pH 7.5, 2.5 mM MgCl₂, 2.5 mM KCl, 2%DMSO.

Reactions were allowed to proceed for 4 hours at 30° C. and terminatedby the addition of 50 mM EDTA (10 μL). After incubating for at least 15minutes, plates were read on a Packard NXT Topcount or AmershamLEADseeker multimodality imaging system.

Cell lines. The cell lines used to evaluate compounds consist of a humanhepatocyte derived cell line (Huh-7) that constitutively expresses agenotype 1a or 1b HCV replicon containing a Renilla luciferase reportergene. These cells were maintained in Dulbecco's modified Eagle medium(DMEM) containing 10% FBS, 100 U/mL penicillin/streptomycin and 1.0mg/mL G418.

HCV Replicon Luciferase Assay.

To evaluate compound efficacy, HCV replicon cells were seeded in 96-wellplates in DMEM containing 10% FBS at a cell density of 10⁴/well.Following incubation at 37° C. overnight, compounds serially diluted inDMSO were added to the cell plates. Alternatively, titrated compoundswere transferred to sterile 384-well tissue-culture treated plates andthe plates seeded with 50 μL of cells at a density of 2.4×10³ cells/wellin DMEM containing 4% FCS (final DMSO concentration at 0.5%). After 3days incubation at 37° C., cells were analyzed for Renilla Luciferaseactivity using the EnduRen substrate (Promega cat #E6485) according tothe manufacturer's directions. Briefly, the EnduRen substrate wasdiluted in DMEM and then added to the plates to a final concentration of7.5 μM. The plates were incubated for at least 1 h at 37° C. then readon a TopCount NXT Microplate Scintillation and Luminescence Counter(Packard) or Viewlux Imager (PerkinElmer) using a luminescence program.The 50% effective concentration (EC₅₀) was calculated using theexponential form of the median effect equation whereEC₅₀=100−[(δF_(inh)/δF_(con))×100].

To assess cytotoxicity of compounds, Cell Titer-Blue (Promega) was addedto the EnduRen-containing plates and incubated for at least 4 hrs at 37°C. The fluorescence signal from each well was read using a Cytoflour 400(PE Biosystems) or Viewlux Imager.

Compound EC₅₀ data is expressed as A: <100 nM; B=100-1000 nM; C>1000nM). Representative data for compounds are reported in Table 2.

TABLE 2 EC₅₀ (uM) Cmpd# Structure 1b 1001

0.1705 B 1002

B 1003

B 1004

0.0477 A 1005

B 1006

1.2100 C 1051

C 1052

0.7888 B 1053

B 1054

C 1055

C 1056

8.3380 C 1057

C 1058

C 2001

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

We claim:
 1. A compound of formula I, including pharmaceuticallyacceptable salts thereof:

wherein n, m are independently 0 or 1; R¹ is methyl; R² is phenyl thatis independently substituted with 0-2 halo or methoxy or is parasubstituted with W—Ar¹; W is —O— or —NH—; Ar¹ is phenyl orpara-halophenyl; R³ is hydrogen, halo, or alkyl; R⁴, R⁵, R⁶ areindependently selected from hydrogen, halo, alkyl, haloalkyl,hydroxyalkyl, alkoxyalkyl, alkoxy, hydroxyalkyloxy, alkoxyalkyloxy,COOR¹¹ and CON(R¹²)(R¹³); R¹¹ is hydrogen, alkyl or cycloalkyl with 0-3substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; R¹²,R¹³ are each independently hydrogen, alkyl or cycloalkyl with 0-3substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; orR¹² and R¹³ can form a ring by joining two atoms, one from each of R¹²and R¹³; R¹⁴ is hydrogen, alkyl or cycloalkyl with 0-3 substituentsselected from halo, hydroxyl, alkoxy, and haloalkoxy; R⁷ is hydrogen,alkyl, halo, N(R²¹)(R²²), or alkylsulfonyl; R²¹ and R²² areindependently hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, alkylsulfonyl,or alkylsulfonylalkyl; or N(R²¹)(R²²) taken together is azetidinyl,pyrrolidinyl, piperidinyl, or piperazinyl, and is substituted with 0-2substituents selected from alkyl, hydroxyalkyl, and hydroxy; X, Y areindependently selected from CR³¹R³², CO, O, NR³³, NCN, S, SO, and S(O)₂;R³¹, R³² are each independently alkyl or cycloalkyl, further substitutedwith 0-3 substituents; or R³¹ and R³² can form a ring by joining twoatoms, one from each of R³¹ and R³²; R³³ is hydrogen, alkyl, cycloalkyl,or S(O)₂R¹¹ with 0-3 substituents selected from halo, haloalkoxy, OR¹¹,NR¹²R¹³, COOR¹¹, CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³; Zis an alkylene or alkenylene chain containing 0-12 groups selected fromthe group consisting of O, NR⁴¹, S, S(O), S(O)₂, C(O), C(O)O, C(O)NR⁴¹,C(S)NR⁴¹, O—C(O)NR⁴¹, NR⁴¹C(O)NR⁴², and Ar², provided that any O or Satom does not directly bond to another O or S atom, such that ring A is11-24 membered; and further wherein the alkylene or alkenylene chain issubstituted with 0-6 substituents selected from alkyl, cycloalkyl, halo,alkoxy, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³, COOR¹¹,CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, NR¹⁴CONR¹²R¹³, and Ar³; R⁴¹, R⁴² ishydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo,haloalkoxy, OR¹¹, NR¹²R¹³, COOR¹¹, CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³,and NR¹⁴CONR¹²R¹³; Ar² is phenyl, 5-membered heteroaryl or 6-memberedheteroaryl, and is substituted with 0-3 substituents selected fromcyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂,NR¹²R¹³, COOR¹¹, CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³;Ar³ is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and issubstituted with 0-3 substituents selected from cyano, halo, alkyl,cycloalkyl, haloalkyl, OH, OR¹¹, haloalkoxy, NH₂, NR¹²R¹³,COOR¹¹CONR¹²R¹³, S(O)₂R¹¹, S(O)₂NR¹²R¹³, and NR¹⁴CONR¹²R¹³.
 2. Acompound of claim 1 wherein n and m are each
 1. 3. A compound of claim 2wherein R² is phenyl substituted with halo.
 4. A compound of claim 3wherein halo is fluoro.
 5. A compound of claim 1 wherein R³, R⁴, R⁶ andR⁷ are each hydrogen.
 6. A compound of claim 1 wherein X and Y are eachNR³³.
 7. A compound of claim 6 wherein R³³ is hydrogen or S(O)₂CH₃.
 8. Acompound of claim 1 wherein A is at least a 12-member ring.
 9. Acompound of claim 8 wherein A is at least a 15-member ring.
 10. Acompound of claim 9 wherein A is at least a 17-member ring.
 11. Acompound of claim 10 wherein A is at least a 19-member ring.
 12. Acompound, including pharmaceutically acceptable salts thereof, which isselected from the group of:


13. A composition comprising a compound of claim 1 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.
 14. Acomposition comprising a compound of claim 12 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.
 15. Amethod of treating hepatitis C infection comprising administering atherapeutically effective amount of a compound of claim 1 to a patient.